Project Summary Nitric Oxide Signaling Chemistry at Non-Heme Sites Nitric oxide (NO) is a powerful signaling molecule with far reaching effects. It plays numerous roles in many disparate areas of human biology, from vasodilation in the cardiovascular system to host defense against microbial pathogens. Although NO donor drugs have long served as therapeutic agents for cardiovascular disease, the discrete molecular mechanisms connected to NO signaling are not well understood. Adding to the complexity of NO biology, molecular relatives of NO such as S-nitrosothiols (RSNOs), nitrite (NO2-) and nitroxyl (HNO) can also exhibit NO-like behavior. Thus, an understanding of the discrete mechanistic pathways by which these species form, interconvert, and react with biologically relevant molecular sites is crucial for the rational development of therapeutics to target NO signaling pathways. For instance, several neurodegenerative diseases are linked to aberrant protein S-nitrosylation, underscoring the need to understand the connection between free NO and protein RSNOs. Copper enzymes are integral to the interconversion of NO and its redox congeners in biological systems. Multicopper oxidases such as ceruloplasmin can serve as NO oxidases, storing NO for later use as S-nitrosothiols (RSNOs) and nitrite (NO2-). On the other hand, CuZnSOD catalyzes the loss of NO from RSNOs and plays a role in vasodilation, antiplatelet aggregation, and regulation of intracellular RSNO levels. RSNOs induce the release of Zn2+ ions from metallothioneins (MTs), reversibly inhibit DNA transcription by some zinc fingers, and can S-nitrosate protein thiols. At high concentrations, however, NO is toxic leading to highly reactive NxOy species responsible for nitrosative stress. Employing models inspired by coordination environments typically experienced by Cu and Zn ions in biology, along with simple Lewis acids, we will carefully outline a variety of pathways for the interconversion molecules that participate in NO signaling. We will develop and examine models that take advantage of the CuII/I redox couple to convert NO into long lived, mobile species such as RSNOs and NO2- (Aim 1). We will outline factors that control S-transnitrosation reactions at zinc and as well as at free thiols employing simple Lewis acids (Aim 2). Through careful study of highly reactive, oxidizing [CuIII](?2-O2N2) and [CuII](?2-O2N) species derived from the interaction of NO and NO2- at copper sites, we will demonstrate a wide range of reactivity patterns that may participate in nitrosative stress under hypoxic conditions (Aim 3).